Levels of Heavy Metals in Grapevine Soil and Leaf Samples in Response to Seasonal Change and Farming Practice in the Cape Winelands

Heavy metal toxicity is a major threat to the health of both humans and ecosystems. Toxic levels of heavy metals in food crops, such as grapes, can have devastating effects on plant health and the market value of the produce. Two important factors that may influence the prevalence of heavy metals in grapevines are seasonal change and farming practices. The objectives of this study were (i) to conduct a detailed pioneer screening of heavy metal levels in soils and grapevine leaf tissues in selected wine farms and (ii) to study the influence of season and farming on heavy metal levels in soils and grapevine leaf tissues. Soil and grapevine leaf samples were collected from demarcated areas in selected vineyards in the Cape Winelands region of South Africa. The sampling was conducted in winter and summer from the same sites. The soil and leaf samples were analysed using inductively coupled plasma mass spectrometry (ICP-MS) techniques. The pooled data from the farms practising conventional or organic farming showed that seasonal variation had no significant effect (DF = 1, 22; p > 0.05) on the heavy metal contents in the soil. When the soil data from the winter and summer months were compared separately or pooled, the influence of agricultural practice was well-pronounced in As (DF = 1, 22, or 46; p < 0.05) and Cu (DF = 1, 22, or 46; p <0.05). The agricultural practice greatly influenced (DF = 1, 22; p< 0.05) Cu, As, Cr, and Hg uptake, with little effect on Ni, Co, Cd, and Hg leaf contents. Generally, the heavy metals studied (Cr, Co, Ni, Zn, As, Cd, Hg, and Pb) were substantially below the maximum permitted levels in plant and soil samples, per the recommendations of the WHO and Er indices, respectively. However, moderate contamination of the soils was recorded for Cr, Ni, Zn, and Pb. Remarkably, the Cu levels in the organic vineyard soils were significantly higher than in the conventional vineyards. Furthermore, based on the Igeo index, Cu occurred at moderate to heavy contamination levels.


Introduction
Natural and anthropogenic activities are responsible for the build-up of dangerous levels of heavy metals. Many factors affect the prevalence of heavy metals in grapevines [1]. According to Alagic et al. [2], one of the primary sources of heavy metals, such as lead, chromium, arsenic, zinc, cadmium, copper, and nickel, in soils is agricultural practices. The emissions of heavy metals from rapidly expanding industrial areas, mining tails, leaded gasoline and paint, fertilizers, animal manure, wastewater irrigation, and pesticides may contaminate the soil [3][4][5][6], leading to many environmental problems. Heavy metal toxicity is a major threat to the health of both humans and ecosystems; their accumulation in food crops, including grapes, can have devastating effects on plant health and the market value of the produce [2,7]. Briffa et al. [8] reviewed the toxicological effects of heavy metals on humans, including oxidative stress, liver damage, fever, pneumonia, asthma, brain damage, death, and DNA damage.
The objectives of this study were (i) to conduct a detailed pioneer screening of heavy metal levels in soils and grapevine leaf tissues in selected wine farms and (ii) to study the influence of season and farming on heavy metal levels in soils and grapevine leaf tissues. This study revealed that farming practices influenced heavy metal contamination, especially Cu-its levels in organic vineyard soils were significantly higher than in conventional vineyards. However, generally, eight of the nine heavy metals studied (Cr, Co, Ni, Zn, As, Cd, Hg, and Pb) were substantially below the maximum permitted levels in plant and soil samples.

Experimental Design
Soil samples and grapevine leaves were collected from demarcated areas in selected vineyards in the Cape Winelands region of South Africa. The sampling was conducted in the winter and summer from the same sites. A deliberate effort was made to ensure that vineyards with different cultivation practices (organic, conventional, and mixed cropping) were selected for this study.

Site Characteristics
Six vineyards (sites) located in different regions of the Western Cape were selected for this study: Stellenbosch (A), Eikenbosch (B), Franschhoek (C), Wolseley (D), Robertson (E), and Piketberg (F) (Figure 1). Soils were obtained from vineyards with different cultivation approaches: organic (semi to 100% organic), conventional, and polyculture approaches.

Soil and Leaf Sampling
At each vineyard, four sampling points 200 m apart were randomly selected, and the sampling points were in the middle of the vineyard's location for the points. From each sampling point, one kilogram of soil samples was collected after removing surface debris using a garden spade at a depth of 15-20 cm. The soil samples were placed in separate paper bags. Fresh leaf material (100 g) from randomly selected plants at sampling points that were 200 m apart was placed in a paper bag. A total of 48 soil and 48 leaf samples were collected from 6 vineyards in the Western Cape, South Africa. The sampling sites were geo-referenced (Table 1). The collection of samples from the same sampling points was carried out in two seasons (summer and winter). The soil and the leaf samples were analysed at the ICP-MS & XRF Laboratory, Stellenbosch University. Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful technique for elemental trace analysis and is recommended for ultra-trace metals due to its increased sensitivity [27][28][29][30].

Sample Preparation and Analysis
The samples were air-dried and sieved (2 mm sieve) before testing. The concentrations (units: µg kg −1 or mg kg −1 ) of the major, minor, and trace elements of (ICP-AES and ICP-MS) Cr, Co, Ni, Cu, Zn, As, Cd, Pb, and Hg combined were determined as described by Berg et al. [31] with slight modifications. Portions of about 0.5 g (dry weight of plant samples) and 0.1 g (soil samples) were digested with 8 mL nitric oxide at 150 • C for 6-8 h. After cooling to room temperature, the samples were filtered, and demineralized water was added to a total volume of 50 mL. Calibration standards for the ICP-MS analysis were prepared from multi-element stock solutions (Spec-troscan, Teknolab As, N-1440 Drsbak). The ICP-MS instrument was calibrated with standard solutions of 50 and 250 ng mL −1 . For the major elements, an additional standard of 1000 ng mL −1 was used. All the calibration standards and blanks were matched with the nitric acid concentration of the samples. The certified reference material 1573a (tomato leaves) was used to validate the analytical methods for determining the botanical materials' major, minor, and trace elements. Accuracy and precision for the soil samples were achieved by using internal quality control standards (WQB-1). The result of the digested solution in mg/L obtained from the ICP was multiplied by the dilution factor in the digestion process using the following formula: mg kg −1 = mg L −1 × [(Final volume mL)/(weight of sample g)]. Analyses were performed on a Plasma Quad I ICP-MS instrument. The ICP-MS was equipped with a peristaltic pump (Ismatec Reglo 100) and a Meinhard nebulizer. The permissible limits for heavy metals in edible plants that were published by the World Health Organization [32,33] and the Food Toxics 2023, 11, 193 5 of 13 and Agriculture Organization of the United Nations (FAO) were used as standards for the comparison and classification of heavy metal levels into three categories (low, optimum, and high); the levels for the individual heavy metals are as follows: 0.5 µg g −1 arsenic (As), 0.02 µg g −1 cadmium (Cd), 1.3 µg g −1 chromium (Cr), 0.01 µg g −1 cobalt (Co), 10 µg g −1 copper (Cu), and 0.03 µg g −1 mercury (Hg).

Contamination and Ecological Risk Assessment
Contamination indices were used to evaluate the influence of anthropogenic activities on the accumulation of heavy metals in the farms (geo-accumulation index [I geo ]) and the ecological risks associated with heavy metal levels (contamination factor [C f ] and ecological risks [E r ]). The following formula was used [34,35]: where C n is the measured concentration of metal in the soil and B n is the background value of a metal.
The background values (mg kg −1 ) for Cr (5.82), Cu (2.98), Cd (0.62), Zn (12), Hg (0.15), and Pb (2.99) were from South Africa [36], the value for As (20) was from the Netherlands [37], and the value for Co (18) was from China [38]. To compensate for possible variations in the background values and minor anthropogenic influences, a factor of 1.5 was used [34]. The degree of metal contamination in soils as defined by Muller [39], with seven soil quality levels ranging from 1 (uncontaminated) to 6 (extremely contaminated), was used ( Table 2).
The ecological risk index of each heavy metal was determined using the method developed by Hakanson [40] ( Table 2). The following equations were used [34,40]: where T r is the toxic response factor for each given pollutant, C f is the contamination factor for each heavy metal, C n is the measured level of each heavy metal in the sediment, B n is the background level of each heavy metal, and E r is the ecological risk index. The toxic response factors [40] are: Cr (2), Co (5), Cu (5), Cd (30), Ni (5), Zn (1), As (10), Hg (40), and Pb (5).  [39], and the ecological risk for metal pollution, E r , [40].

Statistical Analysis
Heavy metal concentrations in the soils and leaf tissues obtained during the winter and summer months from each farm were compared using a one-way analysis of variance (ANOVA). The heavy metal concentrations in the soils and leaf tissues obtained from farms with different farming practices were compared using a one-way analysis of variance (ANOVA). SPSS was used to process and analyse the data.

Levels of Heavy Metals in Soil Samples
Three of the farm sites (Sites A, B, and E) that were sampled practise conventional farming, and the other three farms practise organic farming (Sites D, E, and F). Meanwhile, four farm sites had polycultures, three of which were conventional farms. The average concentrations of heavy metals in the soil samples from six study sites in the Cape Winelands are given in Table 3. The mean concentration of heavy metal in the soil was highest for chromium (58.738 ± 2.988 mg kg −1 ), and the lowest was observed for Hg (0.015 ± 0.0002 mg kg −1 ) at site F. The mean concentrations of Cd and Hg in the soil samples are generally low across all the sites.  [33]; *** = sites which also practised polyculture; -= data not available

Effect of Seasonal Variation on Heavy Metal Deposits in the Soil
The seasonal variations in the distribution of some of the selected heavy metals in soil samples from the Cape Winelands are shown in Figure 2. The levels of Cd and Hg in all the vineyards are generally minimal. Site E recorded the lowest levels of heavy metals in the soil samples analysed. The heavy metal contents of the soil did not vary significantly (DF = 1, 6; p > 0.05) between the winter and summer for all the study sites. Furthermore, when the data from the farms that practice conventional or organic farming were pooled, the seasonal variation had no significant effect (DF = 1, 22; p > 0.05) on the heavy metal contents in the soil.

Levels of Heavy Metals in Soil Samples
Three of the farm sites (Sites A, B, and E) that were sampled practise conventional farming, and the other three farms practise organic farming (Sites D, E, and F). Meanwhile, four farm sites had polycultures, three of which were conventional farms. The average concentrations of heavy metals in the soil samples from six study sites in the Cape Winelands are given in Table 3. The mean concentration of heavy metal in the soil was highest for chromium (58.738 ± 2.988 mg kg −1 ), and the lowest was observed for Hg (0.015 ± 0.0002 mg kg −1 ) at site F. The mean concentrations of Cd and Hg in the soil samples are generally low across all the sites.     The impact of agricultural practices (conventional; Sites A, B, and E) and organic practices (sites C, D and F) on heavy metal deposits in the soil is shown in Figure 3. When the soil data from the winter and summer months were compared separately or pooled, the influence of agricultural practice was well-pronounced in As (DF = 1, 22, or 46; p < 0.05) and Cu (DF = 1, 22, or 46; p < 0.05). There were no significant differences in the overall heavy metal deposits in the soil between organic and conventional agricultural practices in both the summer (DF = 1, 16; F = 0.09; p = 0.76) and winter (DF = 1, 16; F = 0.02; F = 0.76). The ecological risk index based on the contamination factors and background levels showed low ecological risk in the vineyards for eight of the nine heavy metals assessed-the E r was below 40, corresponding to low risk (Tables 2 and 4). Meanwhile, the geo-accumulation index (E r < 0) indicated a low level of soil contamination for Co, As, Cd, and Hg (Table 4), and neither season nor farming practice had a significant effect on soil contamination. However, moderate contamination of the soils was recorded for Cr, Ni, Zn, and Pb (

Effect of Agricultural Practice on Heavy Metal Deposits in the Soil
The impact of agricultural practices (conventional; Sites A, B, and E) and organic practices (sites C, D and F) on heavy metal deposits in the soil is shown in Figure 3. When the soil data from the winter and summer months were compared separately or pooled, the influence of agricultural practice was well-pronounced in As (DF = 1, 22, or 46; p < 0.05) and Cu (DF = 1, 22, or 46; p < 0.05). There were no significant differences in the overall heavy metal deposits in the soil between organic and conventional agricultural practices in both the summer (DF = 1, 16; F = 0.09; p = 0.76) and winter (DF = 1, 16; F = 0.02; F = 0.76). The ecological risk index based on the contamination factors and background levels showed low ecological risk in the vineyards for eight of the nine heavy metals assessedthe Er was below 40, corresponding to low risk (Tables 2 and 4). Meanwhile, the geoaccumulation index (Er ˂ 0) indicated a low level of soil contamination for Co, As, Cd, and Hg (Table 4), and neither season nor farming practice had a significant effect on soil contamination. However, moderate contamination of the soils was recorded for Cr, Ni, Zn, and Pb (

Levels of Heavy Metals in Plant Samples
The average concentrations of heavy metals in the plant samples from the six study sites in the Cape Winelands are provided in Table 5. The highest mean concentration of heavy metals in the plant samples was observed for Cu (87.098 ± 19.481 mg/kg) at site D, and the lowest was observed for Cd (0.002 ± 0.0004 mg/kg), also at site D. There were significant (DF = 5, 18; p < 0.05) variations in the heavy metal contents (Cr, Cu, As, Cd, Hg, and Pb) in the plant leaves among the sites.   [33]; *** = sites with evidence of polyculture farming; means with the same lowercase letters (a or b or c) are not significantly different.

Effect of Agricultural Practice on Heavy Metal Uptake by Plant Samples
Leaf samples from eight cultivars of grapevine plants occurring in the farms were analysed. To determine the impact of agricultural practices on heavy metals, pooled data from conventional (A, B and E) and organic (sites C, D and F) farming sites were statistically compared ( Figure 4 and Table 5). The agricultural practice significantly influenced (DF = 1, 22; p < 0.05) Cu, As, Cr, and Hg uptake, with little effect on Ni, Co, Cd, and Hg. Generally, the heavy metals were substantially below the maximum permitted levels in plants.

Discussion
A key finding of this study is that the heavy metal contents in soils and grape leaves are below the maximum allowed concentrations of heavy metals in the leaf samples, based on the recommendations of the WHO [33]. Furthermore, the heavy metal concentrations in the soil for eight of the nine heavy metals posed low ecological risk based on the classification of ecological risk heavy metal pollution (40). This is good news for wine consumers and the wine industry in South Africa as the Cape Winelands is the largest wine-producing region on the African continent [41,42]. In addition, the seasonal change did not significantly influence variations in the heavy metals. However, farming practices influenced the accumulations of As and Cu, suggesting that pesticide application is a more important factor influencing heavy metal contents in the Cape Winelands. Cu contamination levels in organic farm soils had higher Igeo values (2.3-2.7), which corresponded to moderately to heavily contaminated soils compared with those in conventional farms. In addition to the over-dependence on agrochemicals, rapid industrialization and urbanization contribute significantly to heavy metal contamination through the high use of metal, leaded gasoline, paint, and petrochemical waste disposal and atmospheric deposition [43,44].
Cu and As varied significantly between the farms that employed organic and conventional farming practices. These two elements are contained in some well-known pesticides used in the cultivation of grapevines [45]. The levels of As were higher in the

Discussion
A key finding of this study is that the heavy metal contents in soils and grape leaves are below the maximum allowed concentrations of heavy metals in the leaf samples, based on the recommendations of the WHO [33]. Furthermore, the heavy metal concentrations in the soil for eight of the nine heavy metals posed low ecological risk based on the classification of ecological risk heavy metal pollution (40). This is good news for wine consumers and the wine industry in South Africa as the Cape Winelands is the largest wine-producing region on the African continent [41,42]. In addition, the seasonal change did not significantly influence variations in the heavy metals. However, farming practices influenced the accumulations of As and Cu, suggesting that pesticide application is a more important factor influencing heavy metal contents in the Cape Winelands. Cu contamination levels in organic farm soils had higher I geo values (2.3-2.7), which corresponded to moderately to heavily contaminated soils compared with those in conventional farms. In addition to the over-dependence on agrochemicals, rapid industrialization and urbanization contribute significantly to heavy metal contamination through the high use of metal, leaded gasoline, paint, and petrochemical waste disposal and atmospheric deposition [43,44].
Cu and As varied significantly between the farms that employed organic and conventional farming practices. These two elements are contained in some well-known pesticides used in the cultivation of grapevines [45]. The levels of As were higher in the farms that practice conventional farming. This was expected because many insecticides used to control pests in grapevines have arsenic compounds. The application of foliar fungicides in vineyards and orchards can increase the soil concentration of heavy metals, such as copper (Cu) and zinc (Zn), up to the toxicity threshold for fruit trees and cover crops [13]. However, remarkably, the Cu concentrations in the organic vineyards were higher than in the conventional vineyards in the current study. The Cu I geo and E r values in the organic farms were higher relative to the conventional farms and corresponded to moderate to heavy contamination and moderate ecological risk, respectively. Vannini et al. [35] also reported similar findings in agricultural soils of the Valdichiana area, Tuscany, Italy; the C f and I geo indices for Cu were higher than for the other heavy metals, and they attributed those findings to the increased use of Cu-based products. The accumulation of Cu in soil and plant tissues could be influenced by many factors other than pesticides, such as the mineralization of organic matter, microorganisms, and minerals in the rock. It is worth noting that organic amendments such as compost and manure, which are widely used in organic farming, bind with Cu more tightly than other micronutrients [46]. Previous studies have investigated the levels of heavy metals in grapefruits in Spain and China [47,48].
This study showed that the season did not affect the heavy metal levels. The results from previous studies suggest that heavy metal concentrations in soils, rivers, and leaves vary with the season; generally, higher heavy metal concentrations are more prevalent in the dry season than in the rainy season [17,49]. In a study by Okoro et al. [50] on the concentrations of heavy metals in seawater from Cape Town harbour, South Africa, the authors reported that Sn and Cd occurred at higher levels in the summer, while Hg, Pb, and As were more prevalent in the winter. It is worth noting that the Cape Peninsula region has a Mediterranean climate, characterised by hot and dry summers and cold and rainy winters [51].
Although this study only investigated the concentrations of heavy metals in vineyard soils and grapevine leaves, the results are very relevant because the use of Cu-and Znbased pesticides in vineyards can increase the levels of these metals in wines and grapes. In the current study, the geochemical analysis showed that in addition to Cu, the heavy metals Ni, Zn, Cr, and Pb showed moderate soil contamination. In a study conducted in Sri Lanka, Prabaga et al. [11] found that most of the accumulated metals are mainly concentrated in the leaves of the grape tree than in the fruit. A survey carried out on the west coast of the Oristano province (Sardinia, Italy) revealed that cobalt occurred at a greater level than the legal limit in one vineyard, and the long-term use of copper-based fungicides in vineyards does not represent a cause of concern for the studied areas [52]. A study that investigated cadmium, copper, lead, and zinc concentrations in wines and alcohol-containing drinks from Italy, Bulgaria, and Poland revealed that these metals occurred in low concentrations; however, the Cu and Zn concentrations were highest in the Italian wines (Cu = 0.13 ± 0.05 mg L −1 ; Zn = 0.83 ± 0.56 mg L −1 ) and lowest in the Polish products (Cu = 0.04 ± 0.001 mg L −1 ; Zn = 0.18 ± 0.16 mg L −1 ) [53].

Conclusions
Four (Co, As, Cd, and Hg) of the nine heavy metals occurred at very low concentrations in the vineyard soils and posed low contamination and ecological risks. However, moderate contamination of the soils was recorded for Cr, Ni, Zn, and Pb. Notably, the Cu levels in the organic vineyard soils were significantly higher than in the conventional vineyards, which is surprising and requires further investigation because Cu-based pesticides are generally not used in organic farming. The season had no significant influence on heavy metal contamination. This study provides comprehensive baseline data on heavy metals in vineyard soils and grapevine leaves in the Cape Winelands. The findings of this study can be applied when adopting farming practices that promote a reduction in metals and also highlight the need for continuous monitoring of toxic metals, even in organic farming, for healthier agroecosystems.